United States Patent (19) 11) Patent Number: 4,861,760 Mazuel et al. (45) Date of Patent: Aug. 29, 1989

4,517,216 5/1985 Shim ...... 536/1.1 (54) OPHTHALMOLOGICAL COMPOSITION OF 4,563,366 l/1986 Baird et al...... 426/573 THE TYPE WHICH UNDERGOES 4,638,059 1/1987 Sutherland ...... 536/123 LIQUID-GEL PHASE TRANSITION 4,661,475 4/1987 Bayerlein et al...... 514/54 75 Inventors: Claude Mazuel; Marie-Claire 4,717,713 1/1988 Zatz et al...... 514/54 Friteyre, both of Riom, France 4,746,528 5/1988 Prest et al...... 536/1. 73) Assignee: Merck & Co., Inc., Rahway, N.J. FOREIGN PATENT DOCUMENTS 1072413 2/1980 Canada. (21) Appl. No.: 911,606 134649 3/1985 European Pat. Off. . (22 Filed: Sep. 25, 1986 0142426 5/1985 European Pat. Off...... 514/914 30 Foreign Application Priority Data 1312244 6/1962 France . Oct. 3, 1985 (FR) France ...... 85 14689 OTHER PUBLICATIONS 51) Int. Cl." ...... A61K 31/715; A61K 31/70 Jansson et al.; Carbohydrate Research vol. 124:135-139, 52 U.S.C...... 514/54; 514/912; (1983). 514/913; 514/915; 514/944; 536/1.1; 536/114; Crescenzi et al.; Carbohydrate Research, vol. 536/123 149:425-432, (1986). 58 Field of Search ...... 514/54,944, 912, 913, Primary Examiner-Ronald W. Griffin 514/915,954; 536/123, 114, 1.1 Assistant Examiner-Nancy S. Carson (56) References Cited Attorney, Agent, or Firm-William H. Nicholson; Joseph F. DiPrima U.S. PATENT DOCUMENTS 2,441,729 5/1948 Steiner ...... 514/944 57 ABSTRACT 2,935,447 5/1960 Miller et al...... 514/54 The present invention relates to a pharmaceutical com 4,013,792 3/1977 Eichman et al. ... 574/171. position intended for contacting with a physiological 4,039,662 8/1977 Hecht et al...... 514/54 4,136,173 1/1979 Pramoda et al. ... 514/800 liquid characterized in that said composition is intended 4,136,177 1/1979 Lin et al...... 514/913 to be administered as a non-gelled liquid form and is 4,188,373 2/1980 Krezanoski ...... 514/11 intended to gel in situ, this composition containing at 4,326,052 4/1982 Kang et al. . ... 536/123 least one polysaccharide in aqueous solution, of the type 4,326,053 4/1982 Kang et al...... 536/123 which undergoes liquid-gel phase transition gelling in 4,343,787 8/1982 Katz ...... 514/54 situ under the effect of an increase in the ionic strength 4,407,792 10/1983 Schoenwald et al...... 514/912 4,409,205 10/1983 Shively ...... 514/53 of said physiological liquid. 4,474,751 10/1984 Haslam et al...... 424/78 4,503,084 3/1985 Baird et al...... 426/573 8 Claims, 1 Drawing Sheet U.S. Patent Aug. 29, 1989 4,861,760

O 13.5 54. 108 162 216 Sec FIG-1

FIG 2 4,861,760 1. 2 perature at physiological temperatures, which are of the OPHTHALMOLOGICAL COMPOSITION OF THE order of 25 C-36 C. TYPE WHICHUNDERGOES LIQUID-GEL PHASE Similarly, Canadian Patent No. 1,072,413 describes TRANSTION systems containing a therapeutic or other agent (polox amer), the gelification temperatures of which are made The present invention relates to a pharmaceutical higher than room temperature by using additives. composition containing at least one polysaccharide in The thermally gelling systems have many disadvan aqueous solution, of the type which undergoes liquid tages, including the risk of gelling before administration gel phase transition under the effect of an increase in the by an increase in the ambient temperature during pack ionic strength. 10 aging or storage, for example. The pharmaceutical compositions of the invention U.S. Pat. No. 4,474,751 of Merck & Co., relates to are of the type which undergoes liquid-gel phase transi other systems for delivering drugs based on ther tion under the effect of an increase in the ionic strength. mogelification of gels, but these systems require very They are particularly intended for contacting with large amounts of polymers and this is not always well physiological liquids. Thus the transition occurs at the 15 tolerated by the eye. contact, as the physiological liquids have a higher tonic The present invention relates to a pharmaceutical ity than the one of said compositions. composition intended for contacting with a physiolog The compositions of the invention are specially useful ical liquid characterized in that said composition is in for ophtalmic use, but also as injectable form, the tended to be administered as a non-gelled liquid form formed gel having thus the function of slow-release 20 and is intended to gel in situ, this composition contain form, as by intradermic or intramuscular injections, or ing at least one polysaccharide in aqueous solution, of as galenic form intended for contacting with mucous the type which undergoes liquid-gel phase transition membranes. gelling in situ under the effect of an increase in the ionic A large percentage of drugs administered to the eye is strength of said physiological liquid. lost as a result of lacrimal drainage; this applies espe 25 The prefered pharmaceutical composition according cially in the case of a liquid formulation. In effect, as a to the invention is an ophthalmological composition, result of this drainage, only a small fraction of the dose the physiological liquid being the lacrimal fluid. Thus, administered remains in contact with the cornea for a the present invention overcomes these particular prob few minutes, and an even smaller fraction penetrates 30 lems of administering ophthalmic compositions. into the eye. As a matter of fact, the composition, which takes the To overcome this disadvantage, it is known to use form of a liquid before its introduction into the eye, viscous solutions, gels, eye ointments or solid eye in undergoes a liquid-gel phase transition, and hence plants. changes from the liquid phase to the gel phase, once it Progress has been made in the delivery of drugs by 35 is introduced into the eye, as a result of the ionic the use of these galenical forms, especially by using the strength of the physiological fluid which is in this case, solid implants, by means of which it is possible to re the lacrimal fluid. duce greatly the doses of active principle in the formu This new ophthalmological composition is an amaz lation while retaining a therapeutic response equivalent ingly advantageous form for several reasons. In particu to that which would be induced by an eye lotion, the 40 lar, since the presence of lacrimal fluid is required to latter, in addition, needing to be administered more induce gel formation, any accidental spillage of solution frequently. outside of the eye cannot result in gel formation. Fur Some of these implants function by diffusion. Thus, thermore, in contrast to the thermally gelling systems, for example, in the “OCUSERT (R)' system, one an increase in the ambient temperature cannot result in weekly application of an oval lens in the conjunctival 45 the solution gelling during storage. sac enables an active principle to be delivered by diffu Also, the polymer used can form a gel at concentra sion, but this lens has to be removed after use, which is tions 10- to 100- fold lower than those used in systems a source of problems for the patients. involving thermogelification. It is hence very well tol Others function by dissolution, and, in this case, since erated by the eye. the implants are either soluble or autodegradable ("LA 50 Finally, when these compositions contain a pharma CRISERT (R)' system), their duration of action is much ceutically active substance, such a delivery system shorter. makes it possible to achieve great bioavailability of the In all cases, the solid implants possess a major disad product, and concentrations of active principle which vantage in that many patients find it difficult to tolerate are sustained with time, advantages of a slow delivery the introduction into the conjunctival culs-de-sacs of 55 system. the solid object represented by this implant. Furthermore, in the case of already gelled or semi To solve this problem, galenical forms can be used solid compositions, it is not possible to administer them which are liquid at room temperature and assure a semi by volumetric means, especially when they come from solid form at human body temperature. Such delivery a multi-dose container. To administer these in reproduc systems are described in U.S. Pat. No. 4,188,373, which 60 ible quantities, one is then compelled to employ gravi propose the use of "PLURONIC(R) polyols". metric means. These "PLURONIC(R) polyols" are thermally gel The compositions according to the invention have, ling polymers in which the polymer concentration is on the one hand, the advantage of liquid ophthalmic chosen in accordance with the desired liquid-gel transi compositions, namely reproducible and accurate dos tion temperature. 65 ing, by volumetric means, of the active substance, and However, with the commercially available on the other hand the advantages known for the systems "PLURONIC(R)polymers", it is difficult to obtain a gel in rigid or semisolid gel form, relating to the delivery of of suitable rigidity while maintaining the transition tem active substances. 4,861,760 3 4. The composition according to the invention conse The ophthalmic compositions according to the inven quently has neither the disadvantages of losses of active tion can be used as they are in various applications, and, substances characteristic of simple liquid compositions, for example, to maintain adequate hydration of the eye nor the unpleasant aspects of solid implant systems, nor (treatment of dry eye syndrome). finally the difficulties of administration associated with Furthermore, it appears that the ophtalmic composi gelled or semi-solid compositions. tions according to the invention are especially suitable The Applicant Company has demonstrated that aque for administering to the eye any pharmaceutically ac ous polysaccharide solutions, of the type which under tive substance administered for curative and/or diag goes liquid-gel phase transition under the effect of an nostic purposes. Thus, the present invention relates to a increase in the ionic strength, and are especially suitable 10 pharmaceutical composition which contains at least one according to the invention, are solutions of a polysac pharmaceutically active substance for curative or diag charide obtained by fermentation of a microorganism. nostic purposes. Thus, according to the invention, an extracellular By pharmaceutically active substance, there is under anionic heteropolysaccharide elaborated by the bacte stood one or more drugs and/or one or more diagnostic rium Pseudomonas elodea and known by the name gellan 15 agents. Any active substance can be delivered by the gum is preferably used. compositions according to the invention. The active This polysaccharide, manufactured by KELCO & substance is preferably chosen to be soluble in water, CO., is already used as a gelling agent for culture me although some active substances show greater solubility dium and also in food products. The structure of this than others in the aqueous polysaccharide solutions heteropolysaccharide consists of the following tetrasac according to the invention. Furthermore, active sub charide repeating unit: stances can be in suspension or in emulsion (e.g. emul sions of oil droplets, complex lipidic materials, lipo somes) in the aqueous polysaccharide solutions. There 25 fore, the present invention relates to ophthalmic com which may, or may not, be partially 0-acetylated on its positions containing at least one active substance in (3-D-glucopyranose (p3-D-Glcp) residues. solution or suspension or emulsion in the aqueous poly The preparation of such polysaccharides in native saccharide solution. and deacetylated form is described, in particular, in U.S. The prefered pharmaceutically active substance, used Pat. Nos. 4,326,053 and 4,326,052 of MERCK & CO., 30 according to the present invention is timolol or one of Inc. Rahway N.J., and their structure has been de its derivatives. scribed, in particular, by JANSSON & LINDBERG, Timolol can be used alone or in combination with Carbohydr. res. 124 (1983) 35-9. other pharmaceutically active agents. According to the present invention, aqueous solu The present invention relates to the ophthalmic com tions containing about 0.1% to about 2.0% by weight of 35 positions preferably containing about 0.1% to about gellan gum, and especially of the product known by the 2.0% by weight of the polysaccharide described above, tradename Gelrite (R), which is a low acetyl clarified and about 0.001% to about 5% by weight of at least one grade of gellan gum, are viscous at low ionic strength pharmaceutically active substance. but undergo a liquid-gel transition when the ionic The quantities relating to the aqueous gellan gum strength is increased, and this is the case when this 40 solution make it possible to obtain a suitable gel consis aqueous solution is introduced into the eye. tency and to compensate the loss induced by the steril The rigidity of the gel can be modified by adjusting ization procedures used during the process of manufac the polymer concentration. ture of these ophthalmic compositions. The gellan gum product not only has the property of Other additives can also take part in the ophthalmic changing form the liquid to the solid phase when placed 45 compositions according to the invention. These are, in in a medium of higher ionic strength, but it also pos particular, other polymers suitable for topical applica sesses two advantageous additional properties accord tion to the eye, small amounts of acids or bases for ing to the present invention. adjusting the pH to values suitable for administration to In effect, Gelrite (E) in aqueous solution is thixotropic the eye, nonionic tonicity adjusting agents, surfactants, (FIG. 1) and thermoplastic (FIG. 2). 50 agents for controlling bacterial contamination or, for These two properties enable its fluidity to be in example, other additives for solubilization or stabiliza creased by shaking or slightly warming the sample be tion of the active substance, or any other additive which fore administration to the eye. assist in the formulation. FIG. 1 shows the rheology of a 0.6% aqueous solu If necessary, the gel-inducing effect of ionized active tion of Gelrite (R) at 20° C. (shear stress (Pa) versus 55 substances, for example, which are incorporated in the shear rate (Sec-1)). compositions according to the invention, can be neu FIG. 2 shows the shear stress (Pa) versus temperature tralized by adding to the formulation a suitable ion (C) behavior at a constant shear rate of 86 second pair-forming agent. of a 0.6% Gelrite (R) solution, after a 30% dilution: For example, the slight gelling effect induced by (1) in distilled water; adding 0.1 mg/ml of benzalkonium chloride in a Gelri (2) in a simulated tear fluid. te (R) solution according to the invention can be elimi This latter case of FIG. 2 (2) shows the increase in nated by adding a small amount of acetic acid. The viscosity resulting from the dilution of Gelrite (R) in a Applicant Company has in addition demonstrated that simulated lacrimal fluid. Gelrite (E) solutions according to the invention are com Thus, the Applicant Company has demonstrated gel 65 patible with other formulation ingredients such as vari formation in a rabbit's eye following a 20 ul instillation ous buffers and potential ion pair-forming agents. of a solution containing 0.4% by weight of Gelrite (R) As will emerge in the examples, mannitol can be used deionized water. in the compositions according to the invention in order 4,861,760 5 6 to regulate the tonicity of the medium without changing neostigmine, echothiopate iodide, demecarium bro the gelling properties. mide, carbamoyl choline chloride, methacholine, betha Other tonicity adjusting agents can be used, sorbitol nechol, and analogs thereof: or any sugar for example. mydriatics such as atropine, homatropine, scopola For their administration to the eye, the ophthalmic mine, hydroxyamphetamine, ephedrine, cocaine, compositions according to the invention are adminis tropicamide, phenylephrine, cyclopentolate, oxypheno tered in liquid form, by any conventional means for nium, eucatropine, and analogs thereof: delivering drops, such as an eye-dropper or, for exam other drugs used in the treatment of conditions and ple, the so called "OCUMETER(R)' system. lesions of the eyes such as: The compositions according to the invention can be 10 antiglaucoma drugs for example timolol, and espe administered in the usual manner for eye lotions, in the cially its maleic salt and R-timolol and a combination of inferior cul-de-sac of the conjunctiva on the outside of timolol or R-timolol with pilocarpine, as well as many the eye. other adrenergic agonists and/or antigonists: epineph By way of example, a drop of liquid composition rine and an epinephrine complex, or prodrugs such as containing about 25 mg of ophthalmic composition 15 bitartrate, borate, hydrochloride and dipivefrine deriva enables about 0.0025 mg to about 1.25 mg of active tives and hyperosmotic agents such as glycerol, manni substance to be administered. tol and urea: carbonic anhydrase inhibitors such as acet The active substances, or drugs, or diagnostic agents, azolamide, dichlorphenamide, 2-(p-hydroxyphenyl)- used in the pharmaceutical compositions according to thio-5thiophenesulfonamide, 6-hydroxy-2-benzo the invention are preferably suited to the treatment of 20 thiazolesulfonamide; and 6-pivaloyloxy-2-benzo the disease from which the patient is suffering and/or to thiazolesulfonamide the diagnostic method which it is desired to employ. antiparasitic compounds and/or anti-protozoal com For example, if the patient is suffering from glau pounds such as ivermectin, pyrimethamine, trisul coma, the active substance chosen in preferably a beta fapidimidine, clindamycin and corticosteroid prepara blocker such as timolol or one of its derivatives. 25 tions; Toxicological studies prove the good tolerability of compounds having antiviral activity such as acy gellan gums: acute oral toxicity tests in rats show that clovir, 5-iodo-2'-deoxyuridine (IDU), adenosine arabi the lethal dose 50 (LD50) is greater than 5000 mg per kg; noside (Ara-A), trifluorothymidine, and interferon and acute toxicity tests by inhalation show that exposure of interferon-inducing agents such as poly I:C; rats for 4 hours to a nominal concentration of 6.09 mg/l 30 antifungal agents such as amphotericin B, nystatin, does not cause the death of any animal in a group of 10 flucytosine, natamycin and miconazole: animals, which indicates that the lethal concentration 50 anesthetic agents such as etidocaine cocaine, benoxi (LC50) is greater than 6.09 mg/l. nate, dibucaine hydrochloride, dyclonine hydrochlo DRAIZE-type eye irritation tests in rabbits show that ride, naepaine, phenacaine hydrochloride, piperocaine, the product is not regarded as an eye irritant. 35 proparacaine hydrochloride, tetracaine hydrochloride, When these compositions contain an active sub hexylcaine, bupivacaine, lidocaine, mepivacaine and stance, the objective of such a system for delivering the prilocaine: active substance is to achieve great bioavailability of the ophthalmic diagnostic agents, such as: substance and concentrations of this substance which (a) those used to examine the retina such as sodium are sustained with time. fluorescein; The drugs or diagnostic agents which can be adminis (b) those used to examine the conjunctiva, cornea and tered by means of the ophthalmic compositions accord lacrimal apparatus, such as fluorescein and rose ben ing to the invention are, for example: gal; and antibacterial substances such as beta-lactam antibiot (c) those used to examine abnormal pupillary responses ics, such as cefoxitin, n-formamidoylthienamycin and 45 such as methacholine, cocaine, adrenaline, atropine, other thienamycin derivatives, tetracyclines, chloram hydroxyamphetamine and pilocarpine: phenicol, neomycin, carbenicillin, colistin, penicillin G, ophthalmic agents used as adjuncts in surgery, such polymyxin B, Vancomycin, cefazolin, cephaloridine, as alpha-chymotrypsin and hyaluronidase: chibrorifamycin, gramicidin, bacitracin and sulfon chelating agents such as ethylenediaminetetraacetic amides: 50 acid (EDTA) and deferoxamine: aminoglycoside antibiotics such as gentamycin, kana immunosuppressants and anti-metabolites such as mycin, amikacin, sisomicin and tobramycin: methotrexate, cyclophosphamide, 6-mercaptopurine nalidixic acid and its analogs such as norfloxacin and and azathioprine: and combinations of the compounds the antimicrobial combination fluoroalanine/pentizi mentioned above, such as antibiotics/antiinflammato done, nitrofurazones and analogs thereof: 55 ries combinations such as the combination of neomycin antihistaminics and decongestants such as pyrilamine, sulfate and dexamethasone sodium phosphate, and com chlorpheniramine, tetrahydrazoline, antazoline and ana binations concomitantly treating glaucoma, for example logs thereof: a combination of timolol maleate and aceclidine. anti-inflammatories such as cortisone, hydrocorti Generally, the tears produced by the eye dilute the sone, hydrocortisone acetate, betamethasone, dexa 60 active substance and very rapidly deplete the dose of methasone, dexamethasone sodium phosphate, predni active substance administered by conventional liquid sone, methylprednisolone, medrysone, fluorometho solutions. lone, prednisolone, prednisolone sodium phosphate, The compositions containing a polysaccharide in triancinolone, indomethacin, sulindac, its salts and its aqueous solution according to the invention, of the type corresponding sulfides, and analogs thereof: 65 which undergoes liquid-gel phase transition under the miotics and anticholinergics such as echothiophate, effect of an increase in the ionic strength, are diluted pilocarpine, physostigmine salicylate, diisopropyl less rapidly and make it possible to obtain a sustained fluorophosphate, epinephrine, dipivaloylepinephrine, delivery of the active substance dissolved or suspended 4,861,760 7 in the composition. (To this end, the total ionic strength stance incorporated in a composition according to the of the formulation must be kept as low as possible). This invention, a comparative test was performed. prolonged residence time, permitted by the composition The removal of fluorescein from the conjunctival sac according to the present invention, leads to more effec of rabbits after an installation of fluorescein solution, tive levels of concentration of active substance in the 5 either in distilled water or in a vehicle containing 0.6% lacrimal film. Gelrite (R), was observed by far UV radiation. A test which demonstrated the prolonged presence of In the eyes treated with the aqueous solution, no the active substance after instillation in the eye of a fluorescein remains 3 hours after the instillation, composition according to the invention, and also other whereas in the eyes treated with the vehicle containing characteristics and advantages of the present invention, 10 the Gelrite (R), fluorescein is still persisting 5 hours after appear in the Examples and Figures which follow, the instillation. which illustrate the invention (the percentages being given by weight). EXAMPLE 5 EXAMPLE 1 15 Composition for delivering timolol Studies are carried out in vivo to obtain data concern Simple ophthalmic composition ing the timolol bioavailability from the solution l of example 2. The concentration of timolol in aqueous Solution 1 Solution 2 Solution 3 humor of non-anaesthetized Albino Rabbits is valued. Gelrite (R) 0.6% 0.6% 0.2% 20 Single 50 ul Instillations of Gelrite (E) Formulations benzalkonium (Example 2 solution 1) and Timoptic (R) commercial chloride 0.0% 0.005% -- solutions, each Containing 0.25% of timolol are carried mannitol 4% 4% - out for a comparison purpose. The Gelrite (R) Solutions list Wate 100% 100% 100% were Made with 3Different lots of Gelrite (R) Polymers. The obtained results are shown in the following table:

Concentration of Timolol in Aqueous Humor in ug/ml E S.E.M. (N) Time after instillation GELRITE FORMULATION Average in minutes Lot 001 Lot 002 Lot 003 Gelrite TIMOPTIC 30 4.1 - 0.6 (8) 3.0 - 0.3 (20) 3.2 - 0.4 (8) 3.4 - 0.2 (36) 1.1 - 0.1 (20) 60 2.3 - 0.4 (8) 2.9 - 0.3 (20) 3.0 - 0.2 (8) 2.7 -- 0.2 (36) 0.9 -- 0.3 (16) 120 1.1 - 0.2 (4) 1.6 - 0.2 (16) 1.1 - 0.1 (8) 1.3 - 0.03 (28) 04 - 0.05 (8) 180 1.0 - 0.2 (4) 0.8 -- 0.1 (16) 0.6 - 0.05 (8) 0.8 - 0.06 (28) 0.3 - 0.04 (12) Note: S.E.M. - Standard Error of Mean N = Number of eyes tested

EXAMPLE 2. Composition for delivering timolol 40 The invention is not limited to the above examples; the compositions of the invention are also useful for their application in all pharmaceutical compositions, Solution l Solution 2 Solution 3 which are intended for contacting with the physiolog timolol maleate 0.34% 0.65% 0.34% ical liquids. Gelrite (R) 0.6% 0.6% 0.6% 45 Thus, the present invention also concerns the injecta stanium 0.01% 0.01% -- ble compositions, for intradermic or intramuscular in mannitol 4% 4% 4% jections, and external topical compositions which are sufficient water intended for contacting with mucous membranes. to make 100% 100% 100% We claim: 50 1. A liquid aqueous ophthalmological composition comprising 0.1 to 2% by weight of gellan gum which on EXAMPLE 3 administration to the eye changes from a liquid to a gel Composition for delivering dexamethasone phosphate as a result of the ionic strength of the lacrimal fluid. 2. The composition of claim 2 which additionally 55 comprises about 0.001% to 5% by weight of an ophthal Solution i Solution 2 Solution 3 mic pharmaceutically-active substance. dexamethasone 3. The composition of claim 2 wherein the phar phosphate 0.1% 0.05% 0.1% maceutically-active substance is selected from the SRun 0.6% 0.3% 0.6% group consisting of an antibacterial substance and an chloride 0.01% 0.01% 0.01% 60 anti-glaucoma drug. mannitol 4% 4% 4% 4. The composition of claim 3 wherein the antibacte sufficient water rial substance is norfloxacin and the antiglaucoma drug to make 100% 100% 100% is timolol maleate or a carbonic anhydrase inhibitor. 5. The composition of claim 1 wherein the gellan gum EXAMPLE 4 65 is Gelrite.6. The composition of claim 5 which additionally To demonstrate the prolonged presence of the active comprises about 0.001% to 5% by weight of an ophthal substance in the eye, after instillation of the active sub mic pharmaceutically-active substance. 4,861,760 9 10 7. The composition of claim 6 wherein the phar rial substance is norfloxacin and the antiglaucoma drug maceutically-active substance 1s. selected from the is selected from the group consisting of timolol maleate groupantiglaucoma consisting drug. of an antibacterial substance and an and a carbonic anhydrase inhibitor. 8. The composition of claim 7 wherein the antibacte- 5 k : *k : X

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65 UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 4, 86l, 760 DATED : August 29, 1989 INVENTOR(S) : C. Mazuel; M. Friteyre It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below: On the title page: In the Assignee: Change "Merck & Co., Inc." to --Laboratoires Merck Sharp & Dohme-Chibret--.

Signed and Sealed this Twenty-second Day of May, 1990

Attest:

HARRY F. MANBECK, JR. Attesting Officer Commissioner of Patents and Trademarks